1. Technical Field
The present invention generally pertains to the field of modulating G protein-coupled receptors (GPCR) and of identifying and preparing G protein coupled receptor inhibiting compounds.
2. Description of the Background Art
A great number of chemical messengers exert their effects on cells by binding to G protein-coupled receptors. Ligand binding to those receptors is transduced by heterotrimeric G proteins into intracellular responses. Four main classes of G proteins are distinguishable: Gs, Gi, Gq and G12. G protein-coupled receptors (GPCR) include a wide range of biologically active receptors, such as hormone receptors, viral receptors, growth factor receptors, chemokine receptors, sensory receptors and neuroreceptors. These receptors are activated by the binding of ligand to an extracellular binding site and mediate their actions through the various G proteins. The molecular interactions that occur between the receptor and the G protein are fundamental to the transduction of environmental signals into specific cellular responses. The G proteins themselves play important roles in determining the specificity and temporal characteristics of the cellular response to the ligand-binding signal.
In the inactive state, G proteins are heterotrimeric, consisting of one α, one β and one γ subunit, and a bound deoxyguanosine diphosphate (GDP). Receptor-catalyzed guanine nucleotide exchange resulting in deoxyguanosine triphosphate (GTP) binding to the α subunit activates the G protein. Gα-GTP dissociates from the Gβγ subunits, allowing the Gβγ dimer and the Gα-GTP subunit each to activate downstream effectors. Hydrolysis of GTP to GDP deactivates the complex and turns off the cellular response.
G protein-coupled receptors have seven transmembrane helices which form, on the intracellular side of the membrane, the G protein binding domain. Experiments have suggested that activation of the receptor by ligand binding changes conformation of the receptor, unmasking G protein binding sites on the intracellular face of the receptor. The heterotrimeric G protein interacts with GPCR in a multi-site fashion with the major site of contact between them at the carboxyl terminus of the Gα subunit. Hamm et al., Science 241:832–5, 1998; Osawa and Weiss, J. Biol. Chem. 270:31052–8, 1995; Garcia et al., EMBO J. 14:4460–9, 1995; Sullivan et al., J. Biol. Chem. 269:21519–21525, 1994; West et al., J. Biol. Chem. 260:14428–30, 1985.
The carboxyl terminal 11 amino acids are most important to receptor interaction and to the specificity of this interaction, Martin et al., J. Biol. Chem. 271:361–366, 1996; Kostenis et al., Biochemistry 36:1487–1495, 1997, however other regions on Gα also are involved in receptor contact. In addition, portions of the Gβγ dimer have been implicated in GPCR binding. See Onrust et al., Science 275:381–384, 1997; Lichtarge et al., Proc. Natl. Acad. Sci. USA 93:7507–7611, 1996; Mazzoni and Hamm, J. Biol. Chem. 271:30034–30040, 1996; Bae et al., J. Biol. Chem. 272:32071–32077, 1997. The carboxyl terminal amino acid regions of Gα proteins (and other GPCR binding regions of the heterotrimeric G protein) not only provide the molecular basis of receptor-mediated activation of G proteins, but they also play an important role in determining the fidelity of receptor activation. Conklin et al., Nature 363:274–276, 1993; Conklin et al., Mol. Pharmacol. 50:885–890, 1996.
The G-protein complex thus serves a complex role, as an intermediate that relays the signal from receptor to one or more specific effectors, and as a clock that controls the duration of the signal. Hamm and Gilchrist, Curr. Opin. Cell Biol. 8:189–196, 1996. Multiple receptors can activate a single G protein subtype, and in some cases a single receptor can activate more than one G protein, thereby mediating multiple intracellular signals. In other cases, however, interaction of a receptor with a G protein is regulated in a highly selective manner such that only a particular heterotrimer is bound.
Because G proteins and their receptors influence a large number of intracellular signals mediated by a large number of different chemical ligands, considerable potential for modulation of disease pathology exists. Many medically significant biological processes are influenced by G protein signal transduction pathways and their downstream effector molecules. See Holler et al., Cell. Mol. Life Sci. 340:1012–20, 1999. Therefore, G protein-coupled receptors and their ligands are the target for many pharmaceutical products and are the focus of intense drug discovery efforts. Over the past 15 years, nearly 350 therapeutic agents targeting GPCRs have been successfully introduced into the market. Because of the ubiquitous nature of G protein-mediated signaling systems, and their influence on a great number of pathologic states, it is highly desirable to find new methods of modulating these systems.
Most currently available drugs affecting GPCRs act by antagonizing the binding between a G protein-coupled receptor and its extracellular ligand(s). On the other hand, receptor subtype-selective drugs have been difficult to obtain. A drawback to the classical approach of designing drugs to interfere with ligand binding has been that conventional antagonists are ineffective for some GPCRs such as proteinase activated receptors (PAR) due to the unique mechanism of enzymatic cleavage of the receptor and generation of a tethered ligand. In other cases, intrinsic or constitutive activity of receptors leads to pathology directly, thus rendering antagonism of ligand binding moot. For these reasons, alternative targets for blocking the consequences of GPCR activation and signaling are highly desirable.
One potential alternative target for inhibition by new pharmaceuticals has been the receptor-G protein interface on the interior of the plasma membrane. Konig et al., Proc. Natl. Acad. Sci. USA 86:6878–82, 1989; Acharya et al., J. Biol. Chem. 272:651924, 1997; Verrall et al., J. Biol. Chem. 272:6898–902, 1997. The carboxyl terminus of Gα and other regions of the G protein heterotrimer conform to a binding site at the cytoplasmic face of the receptor. Sondek et al., Nature 372:276–9, 1994; Lambright et al., Nature 369:621–8, 1994; Lambright et al., Nature 379:311–9, 1996; Sondek et al., Nature 379:369–74, 1996; Wall et al., Science 269:1405–12, 1996; Mixon et al., Science 270:954–960, 1995. Peptides corresponding to these binding regions or mimicking these regions, can block receptor signaling or stabilize the active agonist-bound conformation of the receptor. Hamm et al., Science 241:832–5, 1988; Gilchrist et al., J. Biol. Chem. 273:14912–9, 1998. For example, in the case of rhodopsin, the rod photoreceptor, the Gα C-terminal peptide, Gα 340–350, stabilizes the receptor in its active metarhodopsin II conformation. Hamm et al., Science 241-832-5, 1988; Osawa and Weiss, J. Biol. Chem. 270:31052–31058, 1995. Similarly, two carboxyl terminal peptides from GαS (354–372 and 384–394), but not the corresponding peptides from Gαi2, evoke high affinity agonist binding to β2-adrenergic receptors and inhibit their ability to activate Gαs and adenylyl cyclase. Rasenick et al., J. Biol. Chem. 269:21519–21525, 1994.
In general, GPCRs require agonist binding for activation. However, modifications to the receptor amino acid sequence can stabilize the active state conformation without the requirement for a ligand. Stabilization by such ligand-independent means is termed “constitutive receptor activation.” Constitutive (or agonist-independent) signaling activity in mutant receptors has been well documented, but only a few GPCRs have been shown to exhibit agonist-independent activity in the wild type (or native) form. For example, native dopamine D1B and prostaglandin EP1b receptors possess constitutive activity (Tiberi and Caron, J. Biol. Chem. 269:27925–27931, 1994; Hasegawa et al., J. Biol. Chem. 271:1857–1860, 1996). A number of GPCRs, for example, receptors for thyroid-stimulating hormone (Vassart et al., Ann. N.Y. Acad. Sci. 766:23–30, 1995), causing disease in humans have been found to be mutated to exhibit agonist-independent activity. Experimentally, several single amino acid mutations have produced agonist independent activity. β2 and α2 adrenergic receptors, for example, mutated at single sites in the third cytoplasmic loop show constitutive activity (Ren et al., J. Biol. Chem. 268:16483–16487, 1993; Samama et al., Mol. Pharmacol. 45:390–394, 1994). In some cases, a large deletion mutation in the carboxy tail or in the intracellular loops of GPCRs has led to constitutive activity. For example, in the thyrotropin releasing hormone receptor a truncation deletion of the carboxyl terminus Nussenzveig et al., J. Biol. Chem. 268:2389–2392, 1993; Matus-Leibovitch et al., J. Biol. Chem. 270:1041–1047, 1995 or a smaller deletion in the second extracellular loop of the thrombin receptor (Nanevicz et al., J. Biol. Chem. 270:21619–21625, 1995) renders the receptor constitutively active.
These finding have led to a modification of traditional receptor theory (Samama et al., J. Biol. Chem. 268:4625–4636, 1993). It is now thought that receptors can exist in at least two conformations, an inactive conformation (R) and an activated conformation (R*), and that an equilibrium exists between these two states that markedly favors R over R* in the majority of receptors. It has been proposed that in some native receptors and in the mutants described above, there is a shift in equilibrium in the absence of agonist that allows a sufficient number of receptors to be in the active R* state to initiate signaling.
Negative antagonism is demonstrated when a drug binds to a receptor that exhibits constitutive activity and reduces this activity. Negative antagonists appear to act by constraining receptors in an inactive state (Samama et al., Mol. Pharmacol. 45:390–394, 1994). Although first described in other receptor systems (Schutz and Freissmuth, J. Biol. Chem. 267:8200–8206, 1992), negative antagonism has been shown to occur with GPCRs such as opioid (Costa and Herz, Proc. Natl. Acad. Sci. USA 86:7321–7325, 1989; Costa et al., Mol. Pharmacol. 41:549–560, 1992), β2-adrenergic (Samama et al., Mol. Pharmacol. 45:390–394, 1994; Pei et al., Proc. Natl. Acad. Sci. USA 91:2699–2702, 1994; Chidiac et al., Mol. Pharmacol. 45:490–499, 1994), serotonin type 2C (Barker et al., J. Biol. Chem. 269:11687–11690, 1994), bradykinin (Leeb-Lundberg et al., J. Biol. Chem. 269:25970–25973, 1994), and D1B dopamine (Tiberi and Caron, J. Biol. Chem. 269:27925–27931, 1994) receptors. That being stated, the concept of a constitutively active receptors offer insights which explain pathophysiologic conditions. For example, a constitutively active receptor in a disease process such as hypertension may no longer be under the influence of the sympathetic nervous system. In hypertension, a constitutively active GPCR may be expressed in any number of areas including the brain, kidneys or peripheral blood vessels. A newly recognized class of drugs (negative antagonists or inverse agonists) which reduce undesirable constitutive activity can act as important new therapeutic agents. Thus, a technology for identifying negative antagonists of both native and mutated GPCRs has important predictable as well as not yet realized pharmaceutical applications. Furthermore, because constitutively active GPCRs are tumorigenic, the identification of negative antagonists for these GPCRs can lead to the development of anti-tumor and/or anti-cell proliferation drugs.
Mutagenesis of this same region of Gαt has identified several specific amino acid residues in this binding region crucial for Gαt activation by rhodopsin. Martin et al., J. Biol. Chem. 271:361–6, 1996. Substitution of three to five carboxyl-terminal amino acids from Gαq with corresponding residues from Gαi allowed receptors which signal exclusively through Gαi subunits to activate the chimeric α subunits and stimulate the Gαq effector, phospholipase C β. Conklin et al., i Nature 363:274–276, 1993; Conklin et al., Mol. Pharmacol. 50:885–890, 1996. All of these studies suggest that Gα carboxyl peptide sequences are responsible for the specificity of the signaling responses of the individual G proteins. There are 16 unique Gα subunits (Gαi1, Gαi2, Gαi3, GαO1, GαO2, GαZ, Gαt, Gαq, Gα11, Gα14, Gα5, Gα12, Gα13, Gα15/16, GαIF and Gαgust) thought to mediate specific interaction with different GPCRs, several hundred of which have been cloned. Thus, peptides corresponding to G protein regions which bind the GPCR could be used as competitive inhibitors of receptor-G protein interactions. Hamm et al., Science 241-832-5, 1988; Gilchrist et al., J. Biol. Chem. 273-14912-9, 1998. Drug discovery approaches which take advantage of this opportunity, however, are not available. Jones et al., Expert Opin. Ther. Patents 9(12): 1641, 1999.
An important aspect of the modern drug discovery process is the identification of potent lead compounds for use in modern high throughput screening assays. One of the major challenges confronting companies using high throughput screening is the difficulty of identifying useful lead compounds from very large combinatorial libraries. When literally hundreds of thousands of compounds are screened, characterizing the compounds which test positive (including false positives) is an expensive and time-consuming process. Hence, a method which can identify potent lead compounds and reduce the number of false positives in the screening process would be very desirable.